CN108165642A - Aedes albopictus and Aedes aegypti CYTB gene identification reagent boxes and its discrimination method based on high-resolution melting curve - Google Patents

Aedes albopictus and Aedes aegypti CYTB gene identification reagent boxes and its discrimination method based on high-resolution melting curve Download PDF

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CN108165642A
CN108165642A CN201810273284.XA CN201810273284A CN108165642A CN 108165642 A CN108165642 A CN 108165642A CN 201810273284 A CN201810273284 A CN 201810273284A CN 108165642 A CN108165642 A CN 108165642A
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aedes
melting curve
aedes aegypti
cytb
aedes albopictus
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CN108165642B (en
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滕新栋
顾长美
郑健
刘海江
陈晓光
姜华
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SHANDONG INTERNATIONAL TRAVEL HEALTHCARE CENTER
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

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Abstract

The present invention relates to a kind of species identification fields, aedes albopictus and Aedes aegypti identification reagent box based on being a kind of melting curve by high-resolution, its main feature is that designing universal primer according to aedes albopictus and Aedes aegypti CYTB gene orders, differentiate aedes albopictus and Aedes aegypti various forms such as adult, ovum, larva and pupa etc. using the high-resolution melting curve reaction system of optimization.Aedes albopictus and Aedes aegypti can be differentiated, easy to operate, speed is fast, PCR product without post processing, the difference that can detect single base, really realize stopped pipe operation.

Description

Aedes albopictus and Aedes aegypti CYTB genes based on high-resolution melting curve Identification reagent box and its discrimination method
Technical field
The invention belongs to biomedicine technical fields, and in particular to the lineae ablicantes based on a kind of melting curve by high-resolution Yellow-fever mosquito and Aedes aegypti identification reagent box.
Background technology
Port Mosquito population identifies main means as Morphological Identification, reflects by having Morphological Identification experience and mosquito class form The Check and Examination of Port quarantine functionary of capability is identified according to medicine mosquito class taxa key.However, Morphological Identification faces very More problems：The mosquito class professional accreditation talent is few, and the personnel education period is long；Experience level, professional standards, the mosquito class of appraiser are complete Property can cause qualification cycle long with factors such as stages of development, and efficiency is low；Some complicated population such as sibling species groups and similar shape population It is difficult to identify；For mosquito class ovum, larva and pupa, to obtain its secured identification feature, it is often necessary to which mosquito class is identified after manually cultivating； It is very big test to port mosquito class appraisal if inspection mosquito class quantity in port is larger；Etc..Thus, port mosquito class mirror It is fixed other than improving quarantine functionary's identification capacity and research and development identification auxiliary system, it is still necessary to development safely, fast and accurately, it is sensitive Identification method, to realize mosquito class Rapid identification.
In recent years, with the development of molecular biology technology, Standard PCR, restriction fragment length polymorphism （RFLP）, randomly amplified polymorphic DNA（RAPD）, single-strand conformation polymorphism（SSCP）, DNA bar code technology equimolecular means Through being applied to Mosquito population identification.However, these identification methods have shortcoming：Conventional detection freeze-draw method sequence, according to Molecular probe more expensive Lai Yu or product sequencing；RFLP identifications mosquito kind not only takes, but also need great amount of samples；RAPD identifies mosquito Kind is quick, but unstable result, and sometimes not reproducible；SSCP can only will be determined as the method for detection mosquito genoid mutation The position of mutation and type need to further be sequenced, and deposition condition requirement is stringenter；The follow-up sequencing of DNA bar code technology Work takes, consumption funds.In view of the deficiency of the above method, in order to safely, fast and accurately, sensitively realize point of Mosquito population Son identification, needs to seek new Molecular Identification means.
Invention content
For the disadvantages described above or Improvement requirement of the prior art, the present invention provides one kind using high-resolution melting curve as base The aedes albopictus and Aedes aegypti identification reagent box of plinth can be new as aedes albopictus and Aedes aegypti discriminating means, it is with monokaryon Thuja acid melting temperature is different and is formed based on different shape melting curve and carries out mosquito class identification, it is easy to operate, speed is fast, it is low into Originally, high-throughput, accurate, the not examined site of result limitation, PCR products are without post-processing, can detect single base The advantages such as difference realize that real stopped pipe operation so as to reduce pollution risk, is very suitable for the analysis of a large amount of samples.
The present invention is realized by following technical scheme：
The present invention devises one group of aedes albopictus and Aedes aegypti diagnostic primers based on high-resolution melting curve, sequence It is as follows：
CYTB-F：5’-GCTTGTTTATTTATTCACGTA-3’.
CYTB-R：5’-AATCCTCCTCAAATTCATTG-3’.
Using above-mentioned detection primer, the present invention a kind of melting curve by high-resolution is provided based on aedes albopictus and Aedes aegypti identification reagent box, it includes such as ingredient：
1）The universal primer of aedes albopictus and Aedes aegypti ATP6 genes
CYTB-F：5’-GCTTGTTTATTTATTCACGTA-3’.
CYTB-R：5’-AATCCTCCTCAAATTCATTG-3’.
2）PCR mixed liquors：FastStart Taq archaeal dna polymerases, reaction buffer, dNTP mixed liquors, ResoLight satisfy And fluorescent dye.
3）Aedes albopictus and Aedes aegypti genome.
4）ddH2O。
5）25 mM MgCl2。
Wherein, the reaction buffer is PCR buffer (10X) (PCR reaction buffers), is that this field is common Commercialization buffer solution.
The present invention also provides the aedes albopictus and Aedes aegypti discrimination method based on a kind of melting curve by high-resolution, It is characterized in that it includes the following steps：
2）The preparation of high-resolution melting curve template aedes albopictus and Aedes aegypti genomic DNA
It is organized using electric homogenizer cryogrinding aedes albopictus and Aedes aegypti.Use TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 kits (TaKaRa companies), with reference to kit explanation Extract aedes albopictus and Aedes aegypti genomic DNA.
3）High-resolution melting curve reaction system：
30 μ L reaction systems include：CYTB-F 0.6 μL（10 μm of working concentrations）, 0.6 μ L of CYTB-R（10 μm of work are dense Degree）, 2 μ L of template, 3 μ L, FastStart Taq archaeal dna polymerases of reaction buffer, 0.5 μ L, dNTP mixed liquor, 2 μ L, MgCl2 3.6 μ L, ResoLight saturated fluorescence dyestuff 1 μ L, ddH2O 16.7 μL。
4）High-resolution melting curve response procedures：
94 DEG C of 5 min of pre-degeneration.94 DEG C of denaturation 30s, 52 DEG C of annealing 30s, 72 DEG C of extension 1min, totally 30 recycle.72 DEG C of extensions 5min。
High-resolution melting curve is directly run after PCR.95 DEG C of denaturation 1min.40 DEG C of cooling 1min.65-95℃ Continuous warming（25 fluorescence signals are collected in 1 DEG C of heating every time）.40 DEG C of cooling 10s.Use 480 Gene of LightCycler Scanning Software softwares analyze high-resolution melting curve.
The present invention a kind of melting curve by high-resolution based on aedes albopictus and Aedes aegypti identification reagent box and Operating method designs universal primer according to aedes albopictus and Aedes aegypti CYTB gene orders, can to aedes albopictus and it is Egyptian she Adult, ovum, larva and the pupa of mosquito are differentiated, compared with existing discriminating means, advantage of the invention is that：
1）The various states of aedes albopictus and Aedes aegypti can be differentiated, such as adult, ovum, larva and pupa state, be not required to Differentiate after hatching.
2）The primer that the present invention designs, high sensitivity, high specificity.
3）It is easy to operate, speed is fast, the same day can provide result.
4）PCR products realize real stopped pipe behaviour without advantages such as post processing, the differences that can detect single base Make so as to reduce pollution risk, be very suitable for the analysis of a large amount of samples.
Description of the drawings
Fig. 1：Aedes albopictus and Aedes aegypti CYTB PCR result figures, wherein, M：DL2000,1 aedes albopictus, 2 it is Egyptian she Mosquito.
Fig. 2：Aedes aegypti CYTB genes relative to aedes albopictus CYTB genes high-resolution melting curve figure.（Horizontal seat Temperature is designated as, ordinate is relative intensity of fluorescence）, wherein the curve for having fluctuation is Aedes aegypti, straight line is aedes albopictus.
Specific embodiment
Below with embodiment, the invention will be further described.
The present embodiment provides the aedes albopictus and Aedes aegypti identification reagent based on a kind of melting curve by high-resolution Box, including following ingredient：
1）The universal primer of aedes albopictus and Aedes aegypti CYTB genes
CYTB-F：5’-GCTTGTTTATTTATTCACGTA-3’
CYTB-R：5’-AATCCTCCTCAAATTCATTG-3’
2）PCR mixed liquors：FastStart Taq archaeal dna polymerases, reaction buffer, dNTP mixed liquors, ResoLight saturations are glimmering Photoinitiator dye
3）Aedes albopictus and Aedes aegypti genome
4）ddH2O
5）25 mM MgCl2
Aedes albopictus and Aedes aegypti discrimination method based on additionally providing a kind of melting curve by high-resolution, by following Step carries out：
1）It is organized using electric homogenizer cryogrinding aedes albopictus and Aedes aegypti.Use TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 kits (TaKaRa companies), with reference to kit explanation Extract aedes albopictus and Aedes aegypti genomic DNA.
2）30 μ L high-resolution melting curve reaction systems include：CYTB-F 0.6 μL（10 μm of working concentrations）, CYTB- R 0.6 μL（10 μm of working concentrations）, 2 μ L of template, 3 μ L, FastStart Taq archaeal dna polymerases of reaction buffer, 0.5 μ L, dNTP mixed liquor 2 μ L, MgCl2 3.6 μ L, ResoLight saturated fluorescence dyestuff 1 μ L, ddH2O 16.7 μL。
3）High-resolution melting curve response procedures：94 DEG C of 5 min of pre-degeneration.94 DEG C of denaturation 30s, 52 DEG C of annealing 30s, 72 DEG C of extension 1min, totally 30 recycle.72 DEG C of extension 5min.High-resolution melting curve is directly run after PCR.95 DEG C of changes Property 1min.40 DEG C of cooling 1min.65-95 DEG C of continuous warming（25 fluorescence signals are collected in 1 DEG C of heating every time）.40 DEG C of coolings 10s.High-resolution melting curve is analyzed using 480 Gene Scanning Software softwares of LightCycler.
4）As a result：Such as Fig. 1 and Fig. 2, aedes albopictus and Aedes aegypti CYTB PCR amplify band, and amplified fragments are long 230bp is spent, gene sequencing is simultaneously compared with NCBI Blast, and comparison result is corresponding aedes albopictus and Aedes aegypti.It utilizes 480 Gene Scanning Software softwares of LightCycler analyze high-resolution melting curve, find lineae ablicantes she The curve difference of mosquito and Aedes aegypti is apparent, can effectively distinguish.
5）Conclusion：The present invention differentiates aedes albopictus and Aedes aegypti using high-resolution fusion curve for the first time, has expanded mosquito class The visual angle of identification.The present invention is also that mosquito kind is reflected using the high-resolution fusion curve based on aedes albopictus and Aedes aegypti CYTB genes A fixed new direction.This kit is easy to operate, speed is fast, PCR products are without post-processing, can detect single base Difference, really realize stopped pipe operation, the various states of aedes albopictus and Aedes aegypti can be differentiated, such as adult, ovum, children The states such as worm and pupa differentiate after not needing to hatching.
Certainly, it is limitation of the present invention that above description, which is not, and the present invention is also not limited to the example above, the art Those of ordinary skill, in the essential scope of the present invention, the variations, modifications, additions or substitutions made should all belong to the present invention Protection domain.
Sequence table
<110>Shandong international travel health care center
<120>Aedes albopictus and Aedes aegypti CYTB gene identification reagent boxes based on high-resolution melting curve and its Discrimination method
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
gcttgtttat ttattcacgt a 21
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
aatcctcctc aaattcattg 20

Claims (3)

1. aedes albopictus and Aedes aegypti diagnostic primers based on high-resolution melting curve, it is characterized in that, sequence is as follows：
CYTB-F：5’-GCTTGTTTATTTATTCACGTA-3’
CYTB-R：5’-AATCCTCCTCAAATTCATTG-3’.
2. aedes albopictus and Aedes aegypti identification reagent box based on high-resolution melting curve, it is characterized in that, it includes Following ingredient：
1）The universal primer of aedes albopictus and Aedes aegypti CYTB genes
CYTB-F：5’-GCTTGTTTATTTATTCACGTA-3’；
CYTB-R：5’-AATCCTCCTCAAATTCATTG-3’；
2）PCR mixed liquors：FastStart Taq archaeal dna polymerases, reaction buffer, dNTP mixed liquors, ResoLight saturations are glimmering Photoinitiator dye；
3）Aedes albopictus and Aedes aegypti genome；
4）ddH2O；
5）25 mM MgCl2。
3. aedes albopictus and Aedes aegypti discrimination method based on a kind of melting curve by high-resolution, it is characterized in that, it is wrapped Include following steps：
1）The preparation of high-resolution melting curve template aedes albopictus and Aedes aegypti genomic DNA
It is organized using electric homogenizer cryogrinding aedes albopictus and Aedes aegypti；Use TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 kits (TaKaRa companies), with reference to kit explanation Extract aedes albopictus and Aedes aegypti genomic DNA；
2）High-resolution melting curve reaction system：
30 μ L reaction systems include：CYTB-F 0.6 μL（10 μm of working concentrations）, 0.6 μ L of CYTB-R（10 μm of work are dense Degree）, 2 μ L of template, 3 μ L, FastStart Taq archaeal dna polymerases of reaction buffer, 0.5 μ L, dNTP mixed liquor, 2 μ L, MgCl2 3.6 μ L, ResoLight saturated fluorescence dyestuff 1 μ L, ddH2O 16.7 μL；
3）High-resolution melting curve response procedures：
94 DEG C of 5 min of pre-degeneration；94 DEG C of denaturation 30s, 52 DEG C of annealing 30s, 72 DEG C of extension 1min, totally 30 recycle；72 DEG C of extensions 5min；
High-resolution melting curve is directly run after PCR；95 DEG C of denaturation 1min；40 DEG C of cooling 1min；65-95 DEG C of continuous liter Temperature（25 fluorescence signals are collected in 1 DEG C of heating every time）；40 DEG C of cooling 10s；Use 480 Gene of LightCycler Scanning Software softwares analyze high-resolution melting curve.
CN201810273284.XA 2018-03-29 2018-03-29 Aedes albopictus and Aedes aegypti CYTB gene identification kit based on high-resolution melting curve and identification method thereof Active CN108165642B (en)

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
CN109321566A (en) * 2018-08-20 2019-02-12 烟台国际旅行卫生保健中心 A method of it expanding the specific primer of aedes albopictus Cytb gene and is sequenced using it
CN109735535A (en) * 2018-08-20 2019-05-10 烟台国际旅行卫生保健中心 A method of it expanding Dulicidae insect Cytb gene-specific primer and is sequenced using it
CN110885890A (en) * 2019-11-29 2020-03-17 广西壮族自治区农业科学院植物保护研究所 HRM primer, kit and method for rapidly identifying Bactrocera cucurbitae and Bactrocera south Asia
CN111334583A (en) * 2020-01-19 2020-06-26 中国检验检疫科学研究院 Method for identifying aedes species based on PCR-MS mass spectrometry scanning

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CN105039364A (en) * 2015-08-14 2015-11-11 珠海国际旅行卫生保健中心 Sequence of standard gene of DNA (deoxyribonucleic acid) barcode of aedes and application thereof

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CN105039563A (en) * 2015-08-14 2015-11-11 珠海国际旅行卫生保健中心 Probe identifying three aedes simultaneously on the basis of gene chip and kit
CN105039364A (en) * 2015-08-14 2015-11-11 珠海国际旅行卫生保健中心 Sequence of standard gene of DNA (deoxyribonucleic acid) barcode of aedes and application thereof

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109321566A (en) * 2018-08-20 2019-02-12 烟台国际旅行卫生保健中心 A method of it expanding the specific primer of aedes albopictus Cytb gene and is sequenced using it
CN109735535A (en) * 2018-08-20 2019-05-10 烟台国际旅行卫生保健中心 A method of it expanding Dulicidae insect Cytb gene-specific primer and is sequenced using it
CN110885890A (en) * 2019-11-29 2020-03-17 广西壮族自治区农业科学院植物保护研究所 HRM primer, kit and method for rapidly identifying Bactrocera cucurbitae and Bactrocera south Asia
CN110885890B (en) * 2019-11-29 2023-11-21 广西壮族自治区农业科学院植物保护研究所 HRM primer, kit and method for rapidly identifying melon fly and Nanya fruit fly
CN111334583A (en) * 2020-01-19 2020-06-26 中国检验检疫科学研究院 Method for identifying aedes species based on PCR-MS mass spectrometry scanning

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